PROJECT SUMMARY/ABSTRACT Significance: Next generation sequencing (NGS) has has transformed the field of genomics and enabled broad adoption of the technology in biological, translational, and most recently clinical research. The proteomics equivalent to NGS has not yet been realized. As such, the goal of this proposal is to develop an innovative and highly-scalable digital peptide sequencing platform to effectively scale and democratize protein analysis by providing a simple robust workflow generating massively parallel peptide sequencing information. Approach: We propose to develop a single molecule digital peptide sequencing platform, using a novel enzymatic cyclic degradation scheme, paralleling Edman degradation except using mild enzymatic steps rather than harsh chemical reagents. Our assay is comprised of several key technology elements: a population of DNA tagged single molecule peptides on beads; N-terminal amino acid (NTAA) labeling with PITC (Edman chemical agent), binding of NTAA recognition agents labeled with DNA encoding tags, writing of the DNA code to a library element attached to the peptide, and enzymatic removal of a single modified NTAA using an engineered Edmanase (the deliverable of this proposal). This assay is cycled for a dozen or more times to generate a DNA library representing peptide sequence information on millions to billions of peptides that is sequenced on a standard NGS instrument. Public Health Impact: In summary, we believe our Next-Gen peptide sequencing assay usher in the era of ?next gen proteomics? effectively transforming biological, translational, and clinical research, much like NGS technology has already done in the genomics space.